BioInvasions Records (2021) Volume 10, Issue 3: 544–554

CORRECTED PROOF

Rapid Communication The “code red” for Balkan vineyards: occurrence of ishidae (Matsumura, 1902) (: Cicadellidae) in

Tatjana Cvrković1,*, Jelena Jović1, Miljana Jakovljević1, Oliver Krstić1, Slavica Marinković1, Milana Mitrović1 and Ivo Toševski1,2 1Department of Plant Pests, Institute for Plant Protection and Environment, Banatska 33, 11080 Zemun, Serbia 2CABI, 1 Rue des Grillons, 2800 Delémont, Author e-mails: [email protected] (TC), [email protected] (JJ), [email protected] (MJ), [email protected] (OK), [email protected] (SM), [email protected] (MM), [email protected] (IT) *Corresponding author

Citation: Cvrković T, Jović J, Jakovljević M, Krstić O, Marinković S, Mitrović M, Abstract Toševski I (2021) The “code red” for Balkan vineyards: occurrence of Orientus (Matsumura, 1902) (Hemiptera: Cicadellidae), known as the ishidae (Matsumura, 1902) (Hemiptera: mosaic , is an invasive alien species native to the Eastern Palaearctic, but Cicadellidae) in Serbia. BioInvasions also widespread and well established on the eastern territory of North America. Records 10(3): 544–554, https://doi.org/10. Since its first detection in Europe in 1998, this polyphagous species has expanded 3391/bir.2021.10.3.04 its distribution area and rapidly spread through central and western European countries, Received: 14 September 2020 inhabiting a wide range of broadleaf trees and shrubs (e.g. Gleditsia triacanthos, Accepted: 8 February 2021 Salix spp., Corylus spp., Acer spp., Betula spp., Populus spp. and Carpinus spp.), Published: 31 March 2021 which are usually present in the surrounding of vineyards. Over the last decade, Handling editor: Wolfgang Rabitch O. ishidae was frequently found within European vineyards and was proven to be Thematic editor: Angeliki F. Martinou able to complete its life cycle on grapevine. Moreover, this leafhopper was shown to be naturally infected with the Flavescence dorée (FDp), a devastating Copyright: © Cvrković et al. disease which causes significant economic losses in the major vine-producing This is an open access article distributed under terms of the Creative Commons Attribution License countries. This study provides data on the occurrence of O. ishidae in Serbia, clearly (Attribution 4.0 International - CC BY 4.0). confirming that it has expanded its distribution range to the Balkan Peninsula which OPEN ACCESS. could lead to establishment of new FDp epidemiological cycles inside the local vineyard regions. As a consequence, there could be an increased negative impact on phytosanitary situation with a continual spread of this vector species and FDp epidemic outbreaks in Southeastern Europe.

Key words: the mosaic leafhopper, invasive alien species, Flavescence dorée phytoplasma, vector

Introduction The mosaic leafhopper Orientus ishidae (Matsumura) (Hemiptera, Cicadellidae) is an invasive alien species which originates from the Eastern Palaearctic with a wide range of distribution, reported from Japan, Korea, Taiwan and the Philippines (EPPO 2015). Outside of its native range, O. ishidae was recorded for the first time on the territory of the USA in the early nineteenth century, where it was introduced probably via import of ornamental plants of Aralia spinosa (Sanders and DeLong 1919). It is a polyphagous species, inhabiting a wide range of broadleaf trees such as

Cvrković et al. (2021), BioInvasions Records 10(3): 544–554, https://doi.org/10.3391/bir.2021.10.3.04 544 Occurrence of Orientus ishidae in Serbia

Figure 1. Current distribution of Orientus ishidae (Matsumura, 1902) in Europe. First records are indicated with year denoted in the circles. Map from d-maps.com (https://d-maps.com/carte.php?num_car=2232&lang=en).

Gleditsia triacanthos, Salix spp., Corylus spp., Acer spp., Betula spp., Populus spp. and Carpinus spp. (Valley and Wheeler 1985; Nickel 2010). The first record from Europe dates back to 1998 from northern (Guglielmino 2005). Since its first detection, O. ishidae has expanded its range and rapidly spread through central and western European countries: Switzerland (Günthart and Mühlethaler 2002), (Nickel and Remane 2003), Slovenia (Seljak 2004), (Holzinger 2009), Czech Republic (Malenovský and Lauterer 2010), (Mifsud et al. 2010), (Koczor et al. 2013), the Netherlands (den Bieman and van Klink 2015), Great Britain (EPPO 2015), (Klejdysz et al. 2017), (Chireceanu et al. 2017) and (Lock 2019) (Figure 1). Due to its association with wild plants in vineyard surroundings in the areas severely affected by devastating diseases of grapevine yellows , the mosaic leafhopper has gained special attention over the last decade and has become a research focus in viticultural regions across Europe (Mehle et al. 2010; Lessio et al. 2016, 2019; Casati et al. 2017). Placing focus on O. ishidae is justifiable, considering that alien invasive hemipteran species play an important role as phytoplasma vectors in new environments (D’Urso et al. 2019) and can even cause epidemic outbreaks (reviewed in Constable 2010). The best example of this is the epiphytotic outbreak of the Flavescence dorée (FD) in European vineyards (reviewed in Jeger et al. 2016).

Cvrković et al. (2021), BioInvasions Records 10(3): 544–554, https://doi.org/10.3391/bir.2021.10.3.04 545 Occurrence of Orientus ishidae in Serbia

The Flavescence dorée phytoplasma (FDp, taxonomic subgroups 16SrV-C and 16SrV-D) is a phloem-limited plant pathogenic bacteria, which causes the most serious phytoplasma quarantine disease of grapevine in European Union (EU) and EPPO region, with severe economic consequences in the major vine-producing countries (Jeger et al. 2016). The principal insect vector of FDp in European vineyards is a leafhopper of North American origin, titanus (Hemiptera: Cicadellidae), which feeds primarily on grapevine, spreading the disease from plant to plant in an epidemic manner (reviewed in Constable 2010). However, the finding of several wild plant taxa carrying FDp-related genotypes (Alnus glutinosa, A. incana, , Ailanthus altissima, Salix spp. and Corylus avellana) indicate an open transmission cycle of the disease (Angelini et al. 2004; Arnaud et al. 2007; Filippin et al. 2009, 2011; Casati et al. 2017; Malembic-Maher et al. 2020). In addition, several studies have revealed that other polyphagous and/or are involved in the dispersal of FDp from wild reservoir plants into vineyards. The native , europaea, is able to transmit FDp from infectious C. vitalba to the grapevine (Filippin et al. 2009; Krstić et al. 2018), while native leafhoppers Oncopsis alni, Allygus modestus and A. mixtus could transmit FDp sourced from Alnus spp. (Maixner et al. 2000; Malembic-Maher et al. 2020). Recently, alien O. ishidae has been identified as an additional serious risk for spreading epidemic genotypes of FDp in Slovenia, Italy, Switzerland and France (Mehle et al. 2010; Gaffuri et al. 2011; Trivellone et al. 2015; Malembic-Maher et al. 2020). Over the last decade, O. ishidae has been frequently found in European vineyards and their vicinity and was proven to complete its life cycle on the grapevine (Lessio et al. 2019). Moreover, it was shown to be naturally infected with FDp (Mehle et al. 2010) and to be able to transmit this phytoplasma to the grapevine after experimentally forced acquisition (Lessio et al. 2016) and among naturally infected European alders (Malembic-Maher et al. 2020). In Serbian vineyards, the FDp epidemic outbreaks and occurrence of S. titanus were recorded for the first time in 2003 (Duduk et al. 2004; Magud and Toševski 2004). The role of D. europaea as a native vector and C. vitalba as an FDp reservoir plant were subsequently evidenced (Filippin et al. 2009), along with Alnus spp. as natural carriers of FDp-related genotypes (Cvrković et al. 2008). Despite all efforts to control the pathogen, FDp nowadays still spreads in Serbian vineyards and affects all grape-growing regions. For this reason and because of the documented importance of O. ishidae as an alternative vector in transmission routes of FDp, the occurrence of this leafhopper has been intensively monitored over the past decade across Serbia. This study clearly shows that O. ishidae has expanded its range to the Balkan Peninsula, indicating a potential negative impact on the phytosanitary situation as a consequence of further spread of this leafhopper and of FDp outbreaks in southeastern Europe.

Cvrković et al. (2021), BioInvasions Records 10(3): 544–554, https://doi.org/10.3391/bir.2021.10.3.04 546 Occurrence of Orientus ishidae in Serbia

Materials and methods During a study of Auchenorhyncha fauna in the wine-growing regions of Serbia, including Central Serbia (Belgrade, Topola), South-East Serbia (Niš, Pirot) and the Provence of Vojvodina in North Serbia (Subotica, Fruška Gora, Vršac), extensive monitoring was undertaken in diverse natural ecosystems, in deciduous trees along river banks, in forest margins and ornamental trees in urban areas where trees and shrubs reported as preferred hosts for O. ishidae were present. The surveys were made every 15 days from July to September between 2013 and 2020. The were collected for 15 minutes at each location, with standard entomological sweep nets and mouth aspirators. Number of adults and nymphs were recorded on sites with noted O. ishidae occurrence.

Material identification All collected specimens which, according to morphological characters, corresponded to O. ishidae were placed in 96% ethanol. Morphological characters of the adults and nymphs were examined under a Leica MS5 stereomicroscope. The genitalia of males were examined and compared with the descriptions provided by Biedermann and Niedringhaus (2004) and Guglielmino (2005). Nymphs were identified according to Valley and Wheeler (1985).

DNA extraction, PCR amplification and sequencing Sixteen specimens, previously identified as O. ishidae based on morphology, were used for a DNA study. Total DNA from individual insects was extracted using the DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer’s instructions. The mitochondrial cytochrome c oxidase subunit I gene (mtCOI) was amplified and sequenced for its generally good resolution allowing a differentiation at species level (Hebert et al. 2003). PCR amplification for the barcode region (658 bp) of the mtCOI was performed using the LCO1490 and HCOd primers (Folmer et al. 1994; Chetverikov et al. 2015). The polymerase chain reactions (PCR) contained High Yield Reaction

Buffer A with MgCl2 (1x), additional 2.25 mM MgCl2, 0.6 mM of each dNTP, 0.5 μM of each primer and 1U of FastGene Taq DNA polymerase (NIPPON Genetics Europe) in a 20 μL final volume. PCR cycles were performed in a Mastercycler ep gradient S (Eppendorf) applying the following thermal steps: 95 °C for 5 min (initial denaturation), 35 cycles at 94 °C for 1 min, 54 °C for 1 min (annealing), 72 °C for 2 min and a final extension at 72 °C for 10 min. PCR amplicons were purified using the QIAquick PCR purification Kit (QIAGEN) according to the manufacturer’s instructions, and sequenced on an automated equipment by Macrogen Europe (Amsterdam, the Netherlands).

Cvrković et al. (2021), BioInvasions Records 10(3): 544–554, https://doi.org/10.3391/bir.2021.10.3.04 547 Occurrence of Orientus ishidae in Serbia

Table 1. Collection sites with records of Orientus ishidae (Matsumura, 1902) in Serbia. No. of specimens Location Date GPS Dominant plant species (males/females/nymphs) 44.286967N Topola 17.06.2015 Salix purpurea 1 (0/1/0) 20.693517E 23.07.2020 44.791317N 20 (8/10/2) New Belgrade 1 riparia, Populus alba, Malus spp., Prunus spp. 1.09.2020 20.376317E 10 (3/7/0) 30.07.2020 44.777367N Vitis riparia, Populus alba, Salix alba, Amorpha 8 (4/3/1) New Belgrade 2 1.09.2020 20.356383E fruticosa 4 (2/2/0) 30.07.2020 44.769517N Vitis riparia, Populus alba, Salix alba, Amorpha 15 (6/6/3) New Belgrade 3 1.09.2020 20.348350E fruticosa 5 (2/3/0) 30.07.2020 44.741733N Vitis riparia, Populus alba, Salix alba, Amorpha 12 (8/3/1) New Belgrade 4 1.09.2020 20.312983E fruticosa 8 (3/5/0)

Figure 2. A typical site with an established population of Orientus ishidae in Serbia, New Belgrade 4, GPS: 44.741733N; 20.312983E with draperies of Vitis riparia on Populus alba, Salix alba and Amorpha fruticosa. Photograph by Ivo Toševski.

A comparison with sequences from the GenBank was carried out using the BLAST analysis (Basic Local Alignment Search Tool; http://blast.ncbi.nlm. nih.gov/Blast.cgi). In addition, sequences of the barcode portion of the mtCOI gene were compared with BOLD database (Barcode of Life Data Systems; https://www.boldsystems.org/), as well.

Results A single specimen of O. ishidae was registered in 2015 on Salix purpurea in Topola, Central Serbia (leg. Valeria Trivellone). However, a further extensive survey revealed no new records of the species in this region. In 2020, between 23–30 July and on 1 September, a total of 82 individuals (36 males, 39 females and 7 nymphs) of O. ishidae were collected in four localities in a woodland area near the Sava River between the municipalities of New Belgrade and Ostružnica (Table 1). At all four collecting sites, Vitis riparia () was a predominant food plant, forming characteristic draperies on silverleaf poplar (Populus alba) and white (Salix alba) (Figure 2).

Cvrković et al. (2021), BioInvasions Records 10(3): 544–554, https://doi.org/10.3391/bir.2021.10.3.04 548 Occurrence of Orientus ishidae in Serbia

Figure 3. Orientus ishidae (Matsumura, 1902) on Vitis riparia, Serbia, New Belgrade 1, 23.07.2020, GPS: 44.791317N; 20.376317E, 75 m, leg. Tatjana Cvrković.

Adults were also collected on wild apple (Malus sylvestris) and cherry plum (Prunus cerasifera) trees (Rosaceae), as well as on indigo bush (Amorpha fruticosa) (Fabaceae). At the collecting sites along the Sava River, the North American leafhopper S. titanus was also recorded on V. riparia. The collected adult specimens were distinguished by their basic brownish grey color, with a characteristic mosaic-like pattern of white-, yellow- and black across the forewings, head, pronotum and scutellum, an orange pattern on the head between the eyes, dark legs with tarsus and tibia orange, except for tibia of the hind pair legs (Figure 3) and the structure of the male genitalia. In addition, the barcoding sequence of mtCOI of Serbian specimens (Acc. No. MT998287) subjected to BLAST analysis (www.ncbi.nlm.nih. gov/BLAST) revealed a 100% identity with a 585 bp sequence of O. ishidae originating from Canada (Acc. No. KR584471).

Discussion The unintentional introduction of alien species by anthropogenic activities is a growing worldwide phenomenon due to globalization, expanding markets and increasing amount of transported plant and food consignments (D’Urso et al. 2019). Phytophagous insects represent one of the most numerous groups of invertebrates introduced into Europe from other continents, mainly from Asia, North America and Africa and cause significant economic losses by feeding on plants or by transmitting diseases (Roques et al. 2009). To date, thirty non-native species have been accidentally introduced into Europe. Most of them belong to the family Cicadellidae which is the most numerous in species. Half of the introduced alien species was reported in the last 20 years, while twelve are well established in the continent (D’Urso et al. 2019).

Cvrković et al. (2021), BioInvasions Records 10(3): 544–554, https://doi.org/10.3391/bir.2021.10.3.04 549 Occurrence of Orientus ishidae in Serbia

The invasive mosaic leafhopper O. ishidae overwinters at the egg stage; eggs are inserted into the bark of woody plants and in this way the species was probably introduced into Europe through imported live plant material (Malenovský and Lauterer 2010; Mifsud et al. 2010). The rapid range expansion through western and central European countries over the last 20 years indicates that O. ishidae is very adaptable and once established, it can rapidly spread. The main reason is probably its polyphagous feeding behavior, which includes woody plants (e.g. willow, hazelnut, hornbeam, walnut, beech, ash, etc.), as well as herbaceous plants (e.g. Urtica dioica and Helianthus annus) which are widely spread in urban, suburban and ruderal habitats and agroecosystems. After its first introduction into Europe, O. ishidae was recorded also in vineyards and their vicinity and the females were capable of laying eggs on grapevine canes and branches, especially in vineyards and wild rootstocks surrounded by hazelnut and hornbeam plants which are one of their preferred hosts (Lessio et al. 2019). In 2015, during the survey on the diversity of potential grapevine yellows phytoplasma vectors in Serbia, a single specimen of O. ishidae was registered in Topola. Further extensive survey performed on the same locality over the following years revealed no additional specimens of the mosaic leafhopper in vineyards and their surroundings, suggesting that its population wasn’t established in this region, or the population size was still very low and undetectable. This indicates that, if established, populations of O. ishidae are still scarce in the region and the species has not colonised Serbian vineyards yet. Based on the experience from other European countries (Lessio et al. 2016, 2019; Casati et al. 2017; Chireceanu et al. 2019), high population densities in localities where this leafhopper is present indicates that its naturalization and rapid range expansion is possible in the next few years. The occurrence of O. ishidae in Serbia confirmed in 2020, as well as its association with wild V. riparia alongside with S. titanus as the main FDp vector, pose a real threat to the wine production in grape growing regions of Serbia and the Balkans. It is interesting to note that during our survey a number of collected specimens of O. ishidae exceeded the number of captured S. titanus, at all collecting sites (data not shown). The common occurrence of O. ishidae and S. titanus on wild or abandoned Vitis plants are of peculiar concern because of documented importance of these plants as an FDp source in new outbreaks at local scales (Rossi et al. 2019). Besides S. titanus, other polyphagous Auchenorrhyncha species have been identified as FDp vectors, able to acquire and transmit FDp-related genotypes from reservoir plants in the areas surrounding vineyards. Based on genotyping of the epidemiologically informative house-keeping map gene, the FDp is comprised of variants within three genetic clusters namely Map-FD1 (16SrV-C, including strain FD70), FD2 (16SrV-D including

Cvrković et al. (2021), BioInvasions Records 10(3): 544–554, https://doi.org/10.3391/bir.2021.10.3.04 550 Occurrence of Orientus ishidae in Serbia

reference strain FD-D) and FD3 (16SrV-C, reference strain FD-C) (Arnaud et al. 2007). The climbing shrub C. vitalba has been proven as a natural reservoir of the primary FD3 genotype of FDp in Italy, Slovenia, Switzerland, Austria, Hungary and in the Balkans (Angelini et al. 2004; Filippin et al. 2009; Casati et al. 2017; Krstić et al. 2018; Reisenzein and Strauss 2019). Additionally, in France, Italy, Slovenia and the Balkans, alder trees (Alnus glutinosa and A. incana) have been confirmed as reservoir plants in which FD2 related genotypes are dominantly present, but in some plants FD1 and FD3 related genotypes were revealed (Arnaud et al. 2007; Cvrković et al. 2008; Mehle et al. 2011; Radonjić et al. 2013; Atanasova et al. 2014), while a vigorous invasive plant species, A. altissima was found infected with the FD3 genotype in Italy and Croatia and FD2 in Slovenia (Filippin et al. 2011; Plavec et al. 2019; Mehle et al. 2019). All these genotypes are transmitted by different vectors. Specifically, the native planthopper D. europaea transmits FD3 from C. vitalba to the grapevine, while the native leafhoppers O. alni, A. modestus and A. mixtus transmit FD1 and FD2 genotypes sourced by European alders (Malembic-Maher et al. 2020). Orientus ishidae has been identified as an additional potential risk for spreading the FD1 and FD2 genotypes in Slovenia, Italy, Switzerland and France (Mehle et al. 2010; Casati et al. 2017; Malembic-Maher et al. 2020). Recently, in the study from southern Switzerland O. ishidae was found infected with FD1 and FD3 related genotypes also found in asymptomatic (Salix spp.) and in asymptomatic uncultivated hazelnut shrubs sampled in a forest close to a FD infected vineyard, suggesting they could be additional hosts and reservoirs of FDp (Casati et al. 2017). Moreover, recent studies have shown that symptoms of decline on cultivated hazelnut trees are associated with FD1 and FD2 related genotypes causing severe damage in hazelnut plantations in Slovenia (Mehle et al. 2019), while it is known that O. ishidae prefers wild hazelnut plants (Lessio et al. 2016, 2019; Casati et al. 2017) and widespread populations of this species infected with FDp were recorded (Mehle et al. 2010). Epidemiological context of phylogenetic relatedness of the mosaic leafhopper to native FDp phytoplasma vectors, i.e., its potential to transmit the FDp epidemic strains (Malembic-Maher et al. 2020), as well as its feeding on diverse phytoplasma plant reservoirs in a new environment could potentially influence already complex FDp cycle and route(s) of transmission (e.g., Jeger et al. 2016; Casati et al. 2017; Malembic-Maher et al. 2020). Thus, the presence of this exotic leafhopper species in the local pathosystems and its potential spread across viticultural regions of the Balkan Peninsula could lead to new epidemiological cycles inside vineyards and cause new outbreaks, which would further complicate already complex epidemiological situation with FDp in vineyards in Serbia.

Cvrković et al. (2021), BioInvasions Records 10(3): 544–554, https://doi.org/10.3391/bir.2021.10.3.04 551 Occurrence of Orientus ishidae in Serbia

Acknowledgements The authors are grateful to Dr Valeria Trivellone (Illinois Natural History Survey, University of Illinois at Urbana-Champaign, USA) for finding and collecting first specimen of O. ishidae in Serbia. This study was funded by Ministry of Education, Science and Technological Development of the Republic of Serbia, grant 451-03-68/2020-14/200010 and partly by the SCOPES program of the Swiss National Science Foundation (IZ73Z0_152414). We also thank the reviewers for their helpful comments and suggestions.

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